Water-soluble viscous polysaccharides, such as, gum arabic, xanthan gum, guar gum and rhamsan gum, have been widely utilized in the food, paint, paper making, cosmetic, medicine, petroleum recovery and similar industries. In recent years, the demand for such polysaccharides has increased, particularly for the most widely used, xanthan gum. Xanthan gum exhibits excellent characteristics, such as, a thickening function, an emulsion stabilizing effect, salt resistance, pH resistance and stability to various enzymes, which has led to increased usage of xanthan gum as an additive.
Xanthan gum may be obtained by aerobically culturing microorganisms of the Xanthomonas genus, for example, X. campestris in an aqueous culture medium having a pH of from 5.5 to 9 and containing at least one carbon source, e.g., glucose, molasses and starch, and the like, a water-soluble nitrogen source, such as, peptone or yeast extract, a magnesium salt, phosphate ions and other trace components. In general, after the cultivation, the culture medium is sterilized, and the desired component is precipitated with an alcohol, such as, ethanol or isopropanol and then dried. Preparative methods of the xanthan gum are described in U.S. Pat. Nos. 3,020,206, 3,251,479, 3,391,060, 3,433,708, 3,594,280, 4,282,321 and 8,659,026.
In addition to X. campestris, other known Xanthomonas bacteria can be used to produce xanthan gum, such as, X. carotate, X. incanae, X. begoniae, X. paravericola, X. translucens, X. vasculorum and X. hederae.
The usual procedure for carrying out such fermentations involves two separate steps. First, the organism to be used is subjected to a relatively small scale culturing step to build up the number or concentration of bacteria. This is usually referred to herein as the seed fermentation or seed culturing step. Thereafter, a portion of this seed culture having a relatively high concentration of bacteria is inoculated into a larger scale vessel containing an appropriate medium for the actual production of xanthan, referred to herein as the production culture or fermentation. Correspondingly, the medium for the seed culture is referred to as the seed culture medium and that for the production culture is referred to as the production culture medium.
It is conventional to purify the fermentation broth containing the xanthan gum product by the use of centrifugal separation or cake filtration to remove undissolved substances such as, bacteria residue and water insoluble unconsumed nitrogen components therein. However, the employment of the centrifugal separation or cake filtration is not practical from the viewpoints of cost and operation, because the fermentation liquid is highly viscous and thus a dilution step using water and a subsequent concentration step are required.
Other purification techniques are known in which the undissolved substances in the fermentation liquid are solubilized by an enzyme treatment. For example, U.S. application Ser. No. 07/990,758 discloses a clarifying process which comprises a continuous treatment with alkaline protease and lysozyme. U.S. Pat. Nos. 3,966,618 and 4,010,071 disclose clarifying methods using an alkaline and neutral protease. E.P. Pat. No. 78,621 has suggested a method using an acidic protease and a neutral protease, and U.S. Pat. No. 4,119,491 has suggested a technique in which after a protease treatment, an aqueous polymer solution is brought into contact with a silicate solid to remove cells from the aqueous polymer solution. Also disclosed are techniques using other materials with protease, for example, an enzyme treatment using polysaccharase and protease in U.S. Pat. No. 4,431,734, a method using an enzyme having a polygalacturonase activity and an enzyme having a protease activity in U.S. Pat. No. 4,904,568, and a method using a composite enzyme having .beta.-1, 3-glucanase activity and protease activity in E.P. 39,962. Also known are purification techniques using other enzymes including a method using an enzyme with a nuclease activity and a purification method using cellulase as suggested in U.S. Pat. No. 4,729,958 and U.S. Pat. No. 4,416,990, respectively. In all of these methods using enzyme treatments, troublesome steps, such as, dilution and concentration as required by the conventional filtration and centrifugal separation purification techniques can be omitted. Thus, these enzymatic purification methods are advantageous from economical and operational viewpoints.
Nitrogen sources which are used for both the seed culture and the production culture may be either water-insoluble or water soluble. Water-insoluble organic nitrogen sources which can be used in the culture medium include meals, such as, distillation dregs disclosed in U.S. Pat. No. 3,000,790, soybean meal disclosed in U.S. Pat. No. 3,335,447, U.K. Patent Nos. 2,012,792 A and 8,115,854, and corn meal described in U.S. Pat. Nos. 3,271,267 and 3,455,786.
Water-soluble inorganic nitrogen sources include ammonium salts, such as, ammonium nitrate, ammonium bromate, ammonium lactate, ammonium hydrochloride, ammonium phosphate, ammonium acetate, ammonium sulfate and urea, which are described in U.S. Pat. Nos. 3,391,060, 4,245,046, 4,282,321 and 4,394,447, U.K. Patent Nos. 2,012,792 and 8,115,855, French Patent No. 7,605,933, E.P. Patent No. 66,961 and Japanese Patent Application Laid-open Nos. 165,798/1983, 92,591/1986, 173,796/1986 and 9,385/1990.
When an undissolved water-insoluble organic substance, such as, a meal, is utilized as the sole nitrogen source, the growth ratio of the bacteria during the seed fermentation culture and the fermentation productivity of the xanthan gum during the production culture are high, and the xanthan gum product exhibits good viscosity effects in aqueous solution. However, the fermentation broth at the end of the fermentation contains about 5 to 10 g/l of the unconsumed water-insoluble nitrogen component, the cellular residue of the bacteria and the like, in addition to about 20 to 50 g/l of the xanthan gum product. In order to produce xanthan gum having high transparency, it is necessary to remove the water-insoluble component derived from the culture medium and the cellular residue of the bacteria. However, even if a lytic enzyme treatment with protease, lysozyme or the like is utilized, the aqueous solution of xanthan gum and the xanthan gum product which is separated and/or extracted from the fermentation liquid exhibits poor transparency.
On the other hand, if a water-soluble inorganic nitrogen source is used for both the seed culture and production culture medium, the bacterial growth lags during both the seed fermentation culture and the production culture. As a result, the productivity and the final production level of xanthan gum in the production culture is undesirably low, e.g., 10 g/l or less. As used herein, the term "productivity" means the amount of xanthan gum produced per hour.
If a water-insoluble organic nitrogen component is used in the seed fermentation medium and a water-soluble inorganic nitrogen component is used in the production medium, the fermentation liquid at the end of the fermentation contains about 3 to 6 g/l of undissolved substances composed of bacterial residue, water-insoluble unconsumed nitrogen component and the like. Therefore, even if an enzyme treatment is used for purification, the aqueous xanthan gum solution obtained and the xanthan product therefrom are not sufficiently transparent.
When a water-soluble inorganic nitrogen component is used in the seed fermentation medium and a water-insoluble organic nitrogen component is used as the nitrogen source in the production medium, the growth rate of the bacteria in the seed fermentation medium is low, so that the bacterial growth in the production culture also lags. As a result, the productivity of the xanthan gum deteriorates. In addition, the fermentation liquid at the end of the fermentation contains about 4 to 8 g/l of undissolved substances composed of bacterial residue, the water-insoluble unconsumed nitrogen component and the like, and consequently, sufficient purification cannot be achieved with a lytic enzyme treatment.
Additional nitrogen sources include aqueous organic nitrogen components of biological extracts, such as, yeast extract, peptone, bouillon, tryputon, malt extract, enzyme-degraded casein, gelatin and soybean whey as well as protein amino acids, such as, glutamic acid, aspartic acid, alanine, proline and threonine, which have been described in U.S. Pat. Nos. 3,427,226, 3,433,708, 3,391,060, 4,119,546, 4,263,399 and 4,375,512, Japanese Patent Publication No. 42,634/1980, Japanese Patent Application Laid-open Nos. 165,798/1983, 58,089/1985, 92,591/1986, 173,795/1986, 86,894/1989 and 218,701/1990. They do not contain any water-insoluble component and consequently, can be used to prepare xanthan gum having excellent transparency. However, they are not practical because of their cost.
The compositions of the conventional culture media which have been described above and their characteristics are set forth in Table 1 (I), (II), and (III),
TABLE 1 (I) ______________________________________ Compositions of Conventional Culture Medium in Fermentation Production of Xanthan Gum Combi- Nitrogen Component in Nitrogen Component in nation Seed Fermentation Medium Production Medium ______________________________________ 1 Insoluble & organic Insoluble & organic 2 Insoluble & organic Water-soluble & inorganic 3 Insoluble & organic Water-soluble & organic 4 Water-soluble & inorganic Insoluble & organic 5 Water-soluble & inorganic Water-soluble & inorganic 6 Water-soluble & inorganic Water-soluble & organic 7 Water-soluble & organic Insoluble & organic 8 Water-soluble & organic Water-soluble & inorganic 9 Water-soluble & organic Water-soluble & organic ______________________________________
TABLE 1 (II) ______________________________________ Growth Ratio Productivity Undissolved of Bacteria in of XG in Substances in Combi- Seed Fermenta- Production Production nation tion Medium Medium Medium ______________________________________ 1 .DELTA. .smallcircle. Many 2 .DELTA. .DELTA. Many 3 .DELTA. .smallcircle. Many 4 x .DELTA. Many 5 x x Few 6 x x Few 7 .smallcircle. .smallcircle. Many 8 .smallcircle. .DELTA. Few 9 .smallcircle. .smallcircle. Few ______________________________________ .smallcircle. : Good, .DELTA.: Medial, and x: Bad.
TABLE 1 (III) ______________________________________ Cost of Combi- Effect of Expression Culture nation Enzyme Treatment Properties of Viscosity Medium ______________________________________ 1 x .smallcircle. .smallcircle. 2 x .smallcircle. .smallcircle. 3 x .smallcircle. x 4 x .DELTA. .smallcircle. 5 .smallcircle. x .DELTA. 6 .smallcircle. .DELTA. x 7 x .smallcircle. x 8 .smallcircle. .DELTA. x 9 .smallcircle. .smallcircle. x ______________________________________ .smallcircle. : Good, .DELTA.: Medial, and x: Bad.
As shown, none of the conventional fermentation techniques, and combinations of a culture medium compositions provide satisfactory productivity levels, purification and transparency levels of the enzyme treatment, viscosity effects, under desirable economical conditions.